Isoprenoids are compounds derived from the five-carbon molecule, isopentenyl pyrophosphate. Investigators have identified over 29,000 individual isoprenoid compounds, with new ones continuously being discovered. Isoprenoids are often isolated from natural products, such as plants and microorganisms, which use isopentenyl pyrophosphate as a basic building block to form relatively complex structures. Vital to living organisms, isoprenoids serve to maintain cellular fluidity and electron transport, as well as function as natural pesticides, to name just a few of their roles in vivo. Furthermore, the pharmaceutical and chemical communities use isoprenoids as pharmaceuticals, nutriceuticals, flavoring agents, and agricultural pest control agents. Given their importance in biological systems and usefulness in a broad range of applications, isoprenoids have been the focus of much attention by scientists.
Terpenoids or terpenes, a class of isoprenoids, are a highly diverse class of natural products and are of particular interest since numerous commercial flavors, fragrances and medicines such as antimalarial and anticancer drugs, are derived from them. Of particular interest is amorpha-4,11-diene, the sesquiterpene olefin precursor to artemisinin, a valuable and powerful antimalarial natural product. Artemisinin and its derivatives are sesquiterpene lactones containing an endoperoxide bridge that is unique among the antimalarial drugs. Artemisinins have been acclaimed as the next generation of antimalarial drugs because they show little or no cross-resistance to existing antimalarials. However, artemisinin is but one example of a large group of terpene-based natural products that have found use in treating human disease (e.g., Taxol, a diterpene extracted from the Pacific Yew, is extremely effective in the treatment of certain cancers; and limonene, a monoterpene, and related derivatives are believed to inhibit farnesylation of the growth promoting protein RAS, and therefore inhibit malignant cell proliferation.
Conventional means for obtaining isoprenoids include extraction from biological materials (e.g., plants, microbes, and animals). In general, these drugs accumulate in very small amounts in these materials. Therefore, the commercial production of these drugs by extraction and purification from plant materials, for example, provide low yields. In addition, extraction of isoprenoids from biological materials may also require toxic solvents. Finally, isoprenoids typically require further derivatization prior to use, which can also affect the overall yield obtained. Therefore, even though many of the isoprenoids are most active when derivatized, the ability to produce the olefin backbone in large quantities in a genetically and metabolically tractable host will still result in less expensive drugs and derivatives that may be more active than the original natural product.
Furthermore, because of the complexity of these molecules, the chemical syntheses of terpenoids are inherently difficult, expensive and produce relatively low yields (See Danishefsky et al. (1996) J. Amer. Chem. Soc. 118:2843–2859; Nicolaou et al. (1997) Angew. Chem. Int. Ed. 36:2520–2524; and Avery et al. (1992) J. Amer. Chem. Soc. 114:974–979). For example, organic synthesis is usually complex since several steps are required to obtain the desired product. Furthermore, these steps often involve the use of toxic solvents, which require special handling and disposal.
Unfortunately, the difficulty involved in obtaining relatively large amounts of isoprenoids has limited their practical use. In fact, the lack of readily available methods by which to obtain certain isoprenoids has slowed down the progression of drug candidates through clinical trials. Furthermore, once an isoprenoid drug candidate has passed the usual regulatory scrutiny, the actual synthesis of the isoprenoid drug may not lend itself to a commercial scale.
Isoprenoids such as terpenoids are produced from the universal precursors isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), which are synthesized by either of two biosynthetic pathways (FIG. 7). Eukaryotes, with the exception of plants, generally use the mevalonate-dependent (MEV) isoprenoid pathway to convert acetyl-CoA to IPP, which is subsequently isomerized to DMAPP. Plants employ both the MEV and the mevalonate-independent or deoxyxylulose-5-phosphate (DXP) pathways for isoprenoid synthesis. Prokaryotes, with some exceptions, employ the DXP pathway to produce IPP and DMAPP separately through a branch point (FIG. 7). See Rohdich et al. (2002) Proc. Natl. Acad. Sci. USA 99:1158–1163). IPP and DMAPP precursors are essential to Escherichia coli for the prenylation of tRNA's (Connolly et al. (1989) J. Bacteriol. 171:3233–3246) and the synthesis of farnesyl pyrophosphate (FPP), which is used for quinone and cell wall biosynthesis.
Based upon an understanding of these pathways, researchers have looked to biosynthetic production of isoprenoids. Some success has been obtained in the identification and cloning of the genes involved in isoprenoid biosynthesis. For example, U.S. Pat. No. 6,291,745 to Meyer et al. describes the production of limonene and other metabolites in plants. Although many of the genes involved in isoprenoid biosynthesis may be expressed in functional form in E. coli and other microorganisms, yields remain relatively low as a result of minimal amounts of precursors, namely isopentenyl pyrophosphate.
Croteau et al. describe in U.S. Pat. No. 6,190,895 the nucleic acid sequences that code for the expression of 1-deoxyxylulose-5-phosphate synthase, an enzyme used in one biological pathway for the synthesis of isopentenyl pyrophosphate. Low yields of isopentenyl pyrophosphate remain, however, since several more enzymes are needed to catalyze other steps in this isopentenyl pyrophosphate biosynthetic pathway. Further, the reference does not address an alternative pathway for isopentenyl pyrophosphate biosynthesis, namely the mevalonate pathway.
Several laboratories have described the engineering of the DXP pathway to increase the supply of isoprenoid precursors needed for high-level production of carotenoids in E. coli (Farmer et al. (2001) Biotechnol. Prog. 17:57–61; Kajiwara et al. (1997) Biochem. J. 324:421–426; and Kim et al. (2001) Biotechnol. Bioeng. 72:408–415). Balancing the pool of glyceraldehyde-3-phosphate and pyruvate or increasing the expression of 1-deoxy-D-xylulose-5-phosphate synthase (dxs), and IPP isomerase (idi) resulted in increased carotenoid build-up in the cell. Though improvements in isoprenoid production were noted, this approach most likely suffers from limitations by the internal control mechanisms that are present in the native host.
Research has also focused on the isolation of genes from Artemisia annua L. involved in artemisinin synthesis in the hope of lowering the cost of artemisinin production by improving the yields from genetically engineered plants (Mercke et al. (2000), Arch. Biochem. Biophys. 381:173–180; Bouwmeester et al. (1999) Phytochem. 52:843–854; Wallaart et al. (2001) Planta 212:460–465; and Chang et al. (2000) Arch. Biochem. Biophys. 383:178–184). The first gene discovered encoded the amorpha-4,11-diene synthase, which converts FPP to amorpha-4,11-diene.
Thus, the current invention is directed toward solving these and other disadvantages in the art by increasing the typically low yields associated with conventional synthesis of isopentenyl pyrophosphate, and isoprenoids. Specifically, the current invention is directed toward the engineered the expression of a synthetic amorpha-4,11-diene synthase gene and the mevalonate isoprenoid pathway from Saccharomyces cerevisiae in E. coli. Since isopentenyl and dimethylallyl pyrophosphates are universal precursors to all isoprenoids, the strains of this invention can serve as platform hosts for the production of any terpenoid compound for which the biosynthetic synthase gene is available.